Stable neural stem cell lines

ABSTRACT

A systematic and efficient method for establishing stable neural stem cell lines and neuronal progenitor lines is described. The resulting cell lines provide robust, simple, and reproducible cultures of human and other mammalian neurons in commercially useful mass quantities while maintaining normal karyotypes and normal neuronal phenotypes.

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/047,352, filed Jan. 14, 2002, which is acontinuation-in-part of U.S. patent application Ser. No. 09/053,414,filed Apr. 1, 1998 (now abandoned) and a continuation of U.S. Ser. No.09/398,897, filed Sep. 20, 1999 (now abandoned) which claims priority toU.S. provisional application 60/101,354, filed Sep. 22, 1998; U.S.patent application Ser. No. 09/053,414 is a continuation of U.S. patentapplication Ser. No. 08/919,580 filed May 7, 1997 (now U.S. Pat. No.6,040,180) and a continuation-in-part of U.S. patent application Ser.No. 08/719,450, filed Sep. 25, 1996 (now U.S. Pat. No. 5,753,506) whichclaims priority from U.S. provisional patent application 60/018,206,filed May 23, 1996, the entirety of each of which is hereby incorporatedby reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application discloses a systematic and efficient method forestablishing stable neural stem cell lines and neuronal progenitorlines. The resulting cell lines provide robust, simple, and reproduciblecultures of human and other mammalian neurons in commercially usefulmass quantities while maintaining normal karyotypes and normal neuronalphenotypes.

2. Description of the Related Art

A developing fetal brain contains all of the cells germinal to the cellsof an adult brain as well as all of the programs necessary toorchestrate them toward the final network of neurons. At early stages ofdevelopment, the nervous system is populated by germinal cells fromwhich all other cells, mainly neurons, astrocytes and oligodendrocytes,derive during subsequent stages of development. Clearly such germinalcells that are precursors of the normal brain development would be idealfor all gene-based and cell-based therapies if these germinal cellscould be isolated, propagated and differentiated into mature cell types.

The usefulness of the isolated primary cells for both basic research andfor therapeutic application depends upon the extent to which theisolated cells resemble those in the brain. Just how many differentkinds of neural precursor cells there are in the developing brain isunknown. However, several distinct cell types may exist:

a unipotential precursor to neurons only (“committed neuronalprogenitor” or “neuroblast”),

-   -   a unipotential precursor to oligodendrocytes only        (“oligodendroblast”), a unipotential precursor to astrocytes        only (“astroblast”),

a bipotential precursor that can become either neurons oroligodendrocytes, neurons or astrocytes, and oligodendrocytes orastrocytes, and

a multipotential precursor that maintains the capacity to differentiateinto any one of the three cell types.

CNS stem cells are multipotential precursor cells with the innateproperty to differentiate into all major cell types of the mammaliancentral nervous system (CNS) including neurons, astrocytes, andoligodendrocytes. The methods for isolation and differentiation of CNSstem cells and the characterization of differentiated cell types havebeen previously described in detail, U.S. Pat. No. 5,753,506 (Johe).Briefly, CNS stem cells are expanded in serum-free, chemically definedmedium containing basic fibroblast growth factor, bFGF, as the solemitogen. The culture condition permits nearly pure populations of CNSstem cells for a long period both as a mass culture and as a clonalculture.

The mitotic capacity of CNS stem cells, however, is finite. With theprevious culture conditions, it had been difficult to expand CNS stemcells beyond about 30 celldoublings at which point a majority of thecells have lost their capacity for neuronal differentiation and furtherexpand as glial progenitors rather than as multipotential stem cells.The mechanism for this limitation is yet unknown.

We hypothesized that mitotic CNS stem cells secrete an autocrine factoror factors which suppress the entry into cell cycle at the G1 phase ofmitosis. This would effectively antagonize the mitogenic actions of bFGFand initiate the differentiation path. Thus, it is a mechanism toself-regulate the proliferation of CNS stem cells and, in vivo, to limitthe generation of neurons and glia during development. Consistent withthis mechanism is the observation that high cell density promptlydifferentiates CNS stem cells even in the presence of bFGF andregardless of the passage time.

Although the 30 cell-doublings yield 10⁹-fold expansion of cells, amethod for further significant expansion of CNS stem cells would be ofsignificant commercial value. Here, we disclose that constitutiveactivation of c-myc protein in CNS stem cells prevents their spontaneousdifferentiation at high cell density, confers resistance to glialdifferentiation, and increases the mitotic capacity over 60cell-doublings. This procedure thus yields more than a 10¹⁸-foldexpansion of CNS stem cells.

SUMMARY OF THE INVENTION

The present application reveals a method for producing stable cell linesof mammalian neural precursor cells in vitro. The method comprises thesteps of preparing a culture of neural precursor cells in a serum-freemedium; culturing the neural precursor cells in the presence of a firstmitogen, where the first mitogen is selected from the group consistingof aFGF, bFGF, EGF, TGFoc and combinations thereof; contacting the cellswith an agent capable of being taken up by the cells and capable ofexpressing a c-myc gene; and further culturing the cells in a mediumcontaining the first mitogen and a second mitogen, where the secondmitogen is selected from the group consisting of aFGF, bFGF, EGF,TGFαserum and combinations thereof, with the proviso that the secondmitogen is other than the first mitogen.

In a preferred embodiment of the method, the c-myc gene is fused withother DNA elements, where the other DNA elements comprise at least oneelement selected from the group consisting of a ligand binding domainfor an estrogen receptor, an androgen receptor, a progesterone receptor,a glucocorticoid receptor, a thyroid hormone receptor, a retinoidreceptor, and an ecdysone receptor.

In another preferred embodiment of the method, the medium containing thefirst mitogen and the second mitogen further comprises a myc-activatingchemical selected from the group consisting of β-estradiol, RU38486,dexamethasone, thyroid hormones, retinoids, and ecdysone.

In a more preferred embodiment of the method, the mammalian neuralprecursor cells are derived from a human. In another more preferredembodiment of the method, the mammalian neural precursor cells arederived from an in vitro culture of pluripotent embryonic stem cells.

The present application also reveals a cell line produced according tothis method. In a preferred embodiment of the cell line, the cellsmaintain a multipotential capacity to differentiate into neurons,astrocytes and oligodendrocytes. In other preferred embodiments of thecell line, the cells maintain a bipotential capacity to differentiateinto neurons and astrocytes or into astrocytes and oligodendrocytes.

In more preferred embodiments of the cell line, the cells maintain aunipotential capacity to differentiate into neurons or into astrocytes.

The present application also reveals a method for producing stableclonal cell lines of mammalian neural precursor cells in vitro. Themethod comprises the steps of preparing a culture of neural precursorcells in a serum-free medium; culturing the neural precursor cells inthe presence of a first mitogen, where the first mitogen is selectedfrom the group consisting of aFGF, bFGF, EGF, TGFα and combinationsthereof; contacting the cells with an agent capable of being taken up bythe cells and capable of expressing a c-myc gene and a selectablemarker; further culturing the cells in a medium containing the firstmitogen and a second mitogen, where the second mitogen is selected fromthe group consisting of aFGF, bFGF, EGF, TGFα, serum and combinationsthereof, with the proviso that the second mitogen is other than thefirst mitogen; and collecting c-myc treated cells and co-culturing themwith feeder cells free of the selectable marker and capable ofsupporting survival of the c-myc treated cells in a medium containingthe first mitogen and the second mitogen, with the proviso that thesecond mitogen is other than the first mitogen.

In a preferred embodiment of this method, the c-myc gene is fused withother DNA elements, where the other DNA elements comprise at least oneelement selected from the group consisting of a ligand binding domainfor an estrogen receptor, an androgen receptor, a progesterone receptor,a glucocorticoid receptor, a thyroid hormone receptor, a retinoidreceptor, and an ecdysone receptor.

In another preferred embodiment of this method, the medium containingthe first mitogen and the second mitogen further comprises amyc-activating chemical selected from the group consisting ofβ-estradiol, RU38486, dexamethasone, thyroid hormones, retinoids, andecdysone.

In a more preferred embodiment of this method the mammalian neuralprecursor cells are derived from a human. In another more preferredembodiment of this method, the mammalian neural precursor cells arederived from an in vitro culture of pluripotent embryonic stem cells.

The present application also reveals a cell line produced by thismethod. In a preferred embodiment of this cell line, the cells maintaina multipotential capacity to differentiate into neurons, astrocytes andoligodendrocytes. In other preferred embodiments of this cell line, thecells maintain a bipotential capacity to differentiate into neurons andastrocytes or into astrocytes and oligodendrocytes.

In more preferred embodiments of this cell line the cells maintain aunipotential capacity to differentiate into neurons or into astrocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Arrangement of pMycER retrovirus plasmid. A linearized EcoR1fragment containing the human c-myc gene fused to the ligand bindingdomain of the human estrogen receptor gene (Eiler et al., 1989, Nature340: 60-68) was ligated downstream of the 5′LTR of pLXSN retroviralexpression plasmid (Clontech). The final construct also contains aselectable marker, the neomycin resistance gene, Neo^(r), under the SV40promoter, P_(SV4O).

FIG. 2. Growth capacity of MycER-modified human CNS stem cells. In orderto measure the growth rate and capacity, a MycER-modified human CNS stemcell line pool (HK18.2) derived from 18-week old human fetal corticaltissue was continuously expanded in culture for approximately 80 days.At each passage (solid circle), the cells were harvested, counted, and afraction replated into new plates. This process was repeated for 12passages. By dividing the increased cell number from the initial seedingdensity to the time of harvest by the duration of the culture perpassage, an approximate doubling time was estimated (open triangle). Thedotted line across the graph represents the averaged doubling time forthe entire culture period. Cumulative expansion of the cells wascalculated by multiplying the multiples of increased cell number at eachpassage and expressed as “Cumulative Fold-Expansion” over the initialcell number at day 0. The initial starting cell number at day 0 was5.0×10⁶ cells.

FIG. 3. Stability of neuronal differentiation of MycER-modified humanCNS stem cells.

A. Unmodified CNS stem cells differentiated and immunostained withanti-MAP2ab antibody;

B. Unmodified CNS stem cells differentiated and immunostained withanti-TH antibody;

C. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-MAP2ab antibody viewed at low magnification;

D. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-MAP2ab antibody viewed at high magnification;

E. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-TH antibody viewed at low magnification;

F. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-TH antibody viewed at high magnification;

G. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-GABA antibody viewed at low magnification;

H. MycER modified human cortical cells at passage 4, differentiated andimmunostained with anti-GABA antibody viewed at high magnification;

I. MycER modified human cortical cells at passage 9, differentiated andimmunostained with anti-MAP2ab antibody viewed at low magnification;

J. MycER modified human cortical cells at passage 9, differentiated andimmunostained with anti-MAP2ab antibody viewed at high magnification;

K. MycER modified human cortical cells at passage 9, differentiated andimmunostained with anti-TH antibody viewed at low magnification; and

L. MycER modified human cortical cells at passage 9, differentiated andimmunostained with anti-TH antibody viewed at high magnification.

FIG. 4. Stability of neuronal differentiation. MycER-modified humancortical cell lines were differentiated at passage 4 and at passage 11.The number of neurons immunostained for MAP2ab or TH proteins werequantified and their proportions over the total cells are reported.

FIG. 5. MycER modified neuronal progenitors.

A. MycER-modified rat striatal progenitors immunostained with anti-tauantibody;

B. Morphology and arrangement of tau+/TuJ1− neuronal progenitors,immunostained with anti-tau antibody;

C. Morphology and arrangement of tau+/TuJ1+ neuronal progenitors,immunostained with anti-tau antibody; and

D. Morphology and arrangement of tau+/TuJ1+ neuronal progenitors of C,immunostained with anti-TuJ1 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Neural cells in culture are highly plastic. Even a brief exposure tosuboptimal culture conditions such as serum can have subtle yetsignificant long-term effects on the phenotype of the cells. Yet, almostall of the reported neural cultures employ serum as the primary sourceof mitogen. We and others have demonstrated that, in order to preservethe intrinsic differentiation potential of stem cells and other cells,it is critical to reduce the exposure of the cells to serum and thatwell-defined growth factors, particularly bFGF and/or epidermal growthfactor, EGF, in a serum-free medium can proliferate a variety ofdifferent cell types in a single culture (Johe, U.S. Pat. No. 5,753,506;Weiss et. al., U.S. Pat. No. 5,851,832).

In the absence of a particular molecular marker for each of the celltypes, isolating potentially thousands of distinct neural cell typesthat may exist in a single culture had not been feasible. In theprevious works, we have described the methods and compositions ofdistinct CNS stem cell populations that give rise to a variety ofdifferent neurons in culture. Here, a reproducible and efficient methodutilizing over-expression of the c-myc gene to stabilize thedifferentiation potentials of neural cells and to isolate stable clonalcell lines is described.

With continuous passage, CNS stem cells gradually lose their capacity todifferentiate into neurons, thus becoming glial progenitors. Theconditions which accelerate this process include high cell densityduring proliferation, poor attachment of the cells on extracellularmatrix coated surface, and exposure to glia-promoting factors such asCNTF (ciliary neurotrophic factor), LIF (leukemia inhibitory factor),BMPs (bone morphogenic factors) and serum. In order to overcome thisinstability of neuronal differentiation capacity of CNS stem cells, wehave introduced into the cells a cellular proto-oncogene, c-myc, whoseactivity can be regulated by the presence or absence of an extracellularmolecule, 3-estradiol.

Human and rat CNS stem cells harboring the fusion gene were grown in thecontinuous presence of mitogens and 13-estradiol in the culture medium.Growth of the cells were significantly more robust, exhibiting fastermitotic rate, resistance to spontaneous differentiation, and muchgreater overall stability during the expansion. The cells showed no signof neoplastic transformation or anomalous growth pattern or morphology.Upon withdrawal of the mitogens and β-estradiol, the cells initiateddifferentiation promptly and gave rise to heterogeneous morphologiescharacteristic of neurons and glia. Neuronal differentiation wasefficient, exhibiting molecular expression patterns, localization ofneurons-specific proteins, and cell morphologies and behaviorsessentially indistinguishable from the parental unmodified CNS stemcells.

The neuronal population consisted of various neurotransmitterphenotypes, including the tyrosine hydroxylase-positive dopaminergicphenotype in 10-20% of the neurons. Such neuronal differentiationcapacity was stable through over 60 cell doublings resulting in at least1×10¹⁸-fold increase in the number of neurons and glia derived from thestem cells. Thus, the genetic modification and the stem cell culturemethod described here enable the stable isolation of practicallyunlimited numbers of CNS stem cells from all regions of the developingmammalian brain, each CNS stem cell clone giving rise to potentiallydistinct neuronal subtypes in unlimited numbers. The result, then, is alibrary of mammalian neurons, including human, with distinctmolecular/genetic repertoires representing the diverse cellularphenotypes of the mature brains.

EXAMPLES

Construction of c-Myc-Estrogen Receptor Expressing Retrovirus

A retroviral vector containing the neomycin-resistance gene under theSV40 promoter was linearized with EcoR1 and ligated with theEcoR1-fragment of the DNA encoding a fusion gene of human c-myc cDNA andhuman estrogen receptor cDNA (Eiler et al., 1989, Nature 340: 60-68).The fusion gene was placed under the regulation of MMLV long-terminalrepeat sequence (LTR). The overall arrangement of the final retroviralvector, pMycER, is shown in FIG. 1.

Generation of a Producer Cell Line

To establish a cell line stably producing the MycER retrovirus, anamphotropic packaging cell line was transfected with pMycER plasmid.Stable clones were selected with G418 (1 mg/ml, Life Technology Inc.,MD) for 4 weeks. Twenty clones were screened for high titer productionagainst Hela cells according to standard procedure. A cell line, MycER.10, with a retroviral titer of 10⁵ pfu/ml as measured by infection ofrat striatal stem cells was selected for subsequent experiments.

Infection of Rat and Human CNS Stem Cells

Rat and human CNS stem cells were prepared according to previouslyreported procedure (U.S. Pat. No. 5,753,506). Passage 1 cells wereplated at 0.5×10⁶ cells per 100 mm plate and grown for three additionaldays in serum-free N2 medium plus 10 ng/ml bFGF. MycER.10 cells weregrown in DMEM/10% fetal bovine serum to 50-75% confluence, subsequentlyrinsed three times with DMEM, and incubated for 4-16 hours in aretrovirus collection medium (IFM). IFM consisted of the standard N2components (25 mg/L human recombinant insulin, 100 mg/L humanapotransferrin, progesterone, putrescene, sodium selenite) in DMEM plus10 ng/ml bFGF and 1 μg/ml human plasma fibronectin (hFN). IFM containingthe retrovirus was clarified by two centrifugations at 1400 rpm and 3000rpm. The supernatant was mixed with fresh N2 at a 1:1 ratio with freshbFGF and hFN at 10 ng/ml and 1 g/ml final concentrations, respectively,and applied to the 50-75% confluent CNS stem cell culture. The infectionperiod was typically 6 hours. Human CNS stem cells were infected for 1-3times over a 2-3 day period to compensate for their slow mitotic rate.Subsequently, the cells were rinsed three times with Ca²⁺-, Mg²⁺-freeHank's balanced saline solution (HBSS), passaged, and further expandedin N2 plus 10 ng/ml bFGF.

Selection of MycER-Expressing CNS Stem Cells

The CNS stem cells with stable incorporation of MycER retrovirus werepassaged 1-2 days after the infection, replated at 0.5×10⁶ cells per 100mm plate, and selected from 2 days after the infection with 0.1-0.2mg/ml G418, pH 7.4. The complete, optimal growth medium (IGM) wascomposed of DMEM/F12 (1:1), 25 mg/L human recombinant insulin, 100 mg/Lhuman apotransferrin, progesterone, putrescene, sodium selenite, 10ng/ml bFGF, 0.2 μM β-estradiol, 0.1 mg/ml G418, and 10 ng/ml EGF or 1%fetal bovine serum. Fresh bFGF (10 ng/ml, final concentration) was addeddaily and medium was changed once every two days. The cells werepassaged at approximately 50-75% confluence by rinsing three times withHBSS and trypsin (1×) treatment. Trypsin activity was stopped by addingsoybean trypsin inhibitor (1 mg/ml final concentration).

Isolation of Clones

At the end of the G418 treatment for 14 days, the cells were passagedand replated approximately 200-1000 cells per 100 mm plate. Within 24hours post plating, well-isolated single cells were marked with 3 mmcircles on the bottom of the culture plate. Sometimes, the completeculture medium was mixed with an equal volume of the medium conditionedby the same cells at high cell density to enhance cell survival. Markedclones were picked with the aid of cloning rings and by trypsintreatment. Individual clones were expanded as the mass culture andstored frozen.

Almost all of the clones generated this way eventually assumed glialmorphology and failed to differentiate into neurons, even though theculture conditions are identical as those for the high density culture.Thus, it became apparent that the MycERmodified cells in the presence ofserum required a relatively high cell density in order to maintain theirnative differentiation potentials and survive. Thus, the cell densitywas maintained in the range of 0.5×10⁶ to 1.0×10⁶ cells per 100 mm plateby supplementing the clonal density of MycER-expressing cells withunmodified primary stem cells. By maintaining antibiotic selection over5-8 days with 0.1 mg/ml G418, the feeder cell population was graduallykilled while permitting local cell density of G418-resistant MycER cellsto gradually rise so as to sustain their optimal growth. The antibioticselection was maintained throughout subsequent expansion to ensure allremaining cells were MycER-modified cells.

In addition to neural stem cells, immature glial cells and matureastrocytes of both human and rat origin were effective. Fibroblasts werealso useful, but more difficult to manage because their rapidproliferation rate and their high tolerance to G418. Neurons may also beuseful, but their post-mitotic nature rendered them much more resistantto G418. Non-mitotic fibroblasts and other non-neural cells which hadbeen gamma-irradiated or treated with mitotic inhibitors such asarabinoside C or mitomycin may also be effective in supporting thec-myc-modified neural cells.

Differentiation and Characterization of the Mitotically Enhanced CNSStem Cells

CNS stem cells stably expressing MycER were differentiated by platingthe cells at 100,000 cell/cm² or higher cell density and replacing thegrowth medium with N2 without bFGF, without serum, and withoutβ-estradiol. Typically, the cells were allowed to differentiate for 6-30days before immunohistochemical analysis.

Results

-   1. Search for Additional Factors Enhancing Mitotic Capacity of CNS    Stem Cells

The doubling time for human CNS stem cells in N2 medium with bFGF as thesole mitogen is approximately 60 hours which is markedly slower than 24hours for rat CNS stem cells under the identical culture conditions.This may be due to a species difference in certain cell-autonomousproperties of the cells such as a difference in DNA replication rate orin other mitotic phases, G1 or G2, of the cell cycle. We investigatedother factors to accelerate the mitotic rate of human CNS stem cells.

Many purified recombinant human growth factors were tested for theability to enhance the mitogenic activity of bFGF. The mitotic rate ofhuman CNS stem cells was assessed by measuring the proportion of thecells which have incorporated the mitotic label, bromodeoxyuridine(BrdU), during a 24-hour period. Each growth factor was supplied dailyto the culture in addition to bFGF. Only the combination of bFGF+EGF andbFGF+TGFα (transforming growth factor-alpha) accelerated beyond thebFGF-induced mitotic rate of the human CNS stem cells. In bothconditions, the doubling time of the cells increased 1.5 times to 40hours over bFGF alone. Combination of bFGF and fetal bovine serum at 1%or at 10% also accelerated the BrdU-incorporation rate of bFGF-inducedmitotic rate of CNS stem cells to a similar rate.

-   2. Resistance of CNS Stem Cells Against Spontaneous Differentiation

Although EGF, TGFα, % FBS, or 10% FBS plus bFGF increased the mitoticrate of human CNS stem cells, even under these conditions, CNS stemcells were susceptible to spontaneous differentiation at near-confluentcell density and also prone to drift toward glial progenitor states withmultiple passages. In order to provide enhanced mitotic capacity andgreater stability to the neuronal differentiation capacity of CNS stemcells, we constructed a retroviral vector expressing a fusion protein ofhuman c-myc and human estrogen receptor genes under the MMLV longterminal repeat (FIG. 1).

Dividing mammalian CNS stem cells were infected by the amphotropicretrovirus with high efficiency and the resulting cells selected bytheir resistance to G418 treatment. The transcriptional activity ofc-myc in the fusion protein (MycER) was regulated by the presence orabsence of the estrogen receptor ligand, β-estradiol, in the culturemedium. Moreover, the promoter activity of the long terminal repeat isshut down during CNS stem cell differentiation into neurons, effectivelyeliminating MycER transcript. Combination of the withdrawal of mitogens,absence of β-estradiol, and limited transcription activity of LTRresulted in an efficient constitutive differentiation of CNS stem cellsin a manner indistinguishable from the unmodified parental cells.

CNS stem cells from various regions and developmental stages of humanfetal brains were infected at passagel with the MycER-expressingretrovirus. Infected cells were selected by G418 resistance and expandedin N2B medium (N2 without phenol red) containing bFGF, 10 ng/ml EGF or1% FBS, and β-estradiol. Expression of MycER itself did not cause asignificant change in the mitotic rate of the cells; however, theaddition of EGF and/or FBS significantly increased the mitotic rate andenhanced the overall stability of the culture. The cells proliferatedrobustly, maintained stable morphologies over many successive passages,and sustained their multipotentiality without spontaneousdifferentiation even at nearly confluent cell density. Upon replacementof the growth medium with N2B without any mitogen and withoutβ-estradiol, the stem cells promptly differentiated to give rise toneurons, astrocytes and oligodendrocytes.

-   3. Expansion Capacity of the Mitotically Enhanced CNS Stem Cell    Lines

To ascertain the extent of the mitotic and differentiative capacity ofthe MycER-modified human CNS stem cell line, the cells were expandedcontinuously for 80 days in culture and through 12 passages since theinfection event. During this period, the cell yield at each passage wasmeasured to quantify the actual arithmetic increase in cell number andto determine the stability of the mitotic rate over time (FIG. 2).Overall, the cells went through approximately 54 doublings whichresulted in 10¹⁵-fold increase in the cell number. The doubling time ofthe cells was remarkably constant at about 40 hours per mitosis, whichis unchanged from that of the parental primary human CNS stem cells(FIG. 2).

The same human CNS stem cell preparation was also subjected toMycER-retrovirus infection and grown in bFGF alone or in bFGF and EGF.In bFGF alone as the mitogen, the MycER-expressing CNS stem cellsexhibited enhanced mitotic capacity over the unmodified cells, but yetshowed far less proliferative capacity than in bFGF+1% FBS. As with theunmodified parent cells, the MycER cells also retained a 60 hourdoubling time in bFGF alone. On the other hand, in bFGF+EGF, the MycERexpressing stem cells displayed increased mitotic rate, increasedmitotic capacity, increased stability of neuronal differentiationcapacity, and were resistant to spontaneous differentiation quitesimilar to the bFGF+1% FBS condition. In the absence of MycERexpression, the same three conditions yielded a similar pattern ofgrowth, but with less stability. Significantly, the bFGF+1% FBScondition, although resulting in more efficient cell growth, inevitablyled to the loss of the neuronal differentiation capacity. Thisdemonstrates that the constitutive c-myc function in these cells issubtle: It provides more stable multipotentiality and enhanced mitoticcapacity but not an overt mitogen-independence or transformation.

These effects of the constitutively active c-myc could also be extendedto CNS stem cells from all regions of rat and human fetal brains.

-   4. Neuronal Differentiation of MycER-Enhanced CNS Stem Cells

The MycER-enhanced CNS stem cells were differentiated by withdrawing themitogens and β-estradiol from the medium and without addition ofexogenous factors. Divergence of neuronal and glial morphologies beganto occur within two days. By the third day, neuronal morphologies wereclearly distinguishable. The neurons continued to mature into fullyfunctional neurons over the next 3-5 weeks.

The differentiated cultures from the MycER-enhanced human CNS stem cellsat different passages were analyzed by immunohistochemistry with avariety of different cell-type specific antibodies. At 10 days ofdifferentiation, approximately 50% of the total cells expressed MAP2c,tau, and tubulin llb proteins, all relatively early markers of neuronaldifferentiation. Approximately 20-30% of the total cells expressed themature markers of neurons, MAP2a and MAP2b proteins. Variousneurofilament antibodies revealed a similar proportion of neurons. Ofthe neurons, approximately 70% were GABA-positive. A similar proportionof neurons was also calretinin-positive. Approximately 10-20% of theneurons expressed tyrosine hydroxylase (TH), the key biosynthetic enzymefor dopamine. All of the immunopositive neurons were of typical neuronalmorphologies and did not co-express the glial marker, GFAP. Thus,MycERenhanced cell lines differentiate to generate a high proportion ofneurons exhibiting various neurotransmitter phenotypes.

The proportions and neurotransmitter phenotypes of the neurons werestable through many successive passages (FIG. 3A-L). Throughout 54 stemcell doublings, there was no degradation of the neuronal differentiationcapacity in both the proportion of neurons as well as in the variousneurotransmitter phenotypes generated.

-   5. Region-Specific Stem Cell Lines

The serum-free culture condition used for isolation of neural stem cellspermitted stable inheritance of regional identities and their relatedneurotransmitter phenotypes through multiple cell divisions. Thisimplies that the stem cells in the culture, although they are uniform intheir ability to differentiate into neurons and glia, may be extremelydiverse. Thus, if each stem cell in the beginning of the culture couldbe immortalized in its native state and if this method was efficient tosample thousands of stem cells in a single dish, then the diverseneuronal phenotypes might be permanently “captured” in the form of celllines.

The genetic modification of neural stem cells with c-myc resulted inrobust, highly reproducible and in a stable cell culture system. Themodification process itself is quite efficient, yielding 5,000 to 50,000independent clones per retrovirus infection over a two day process. Thiscould be easily scaled up by increasing the retrovirus particles orincreasing the number of target cell density, if needed.

In order to ascertain whether over-expression of c-myc has an impact ondifferentiation capacity of neurotransmitter phenotypes, stem cells frommany different regions of the fetal brains of rat and human weremodified by the MycER retrovirus. These regions included cortex, septum,hippocampus, midbrain, hindbrain, striatum, and spinal cord. Multipleexamples of cortical, midbrain, and spinal cord cultures from severaldifferent gestational ages were examined to assess the reproducibilityof the method. In all cases, the resulting pools of independent clonesgenerated highly reproducible ratios of neurons to glia. As expected,morphologies, antigenic profiles of the neurons, and their relativeratios were also distinct from cell lines of one region versus another.

Thus, when several pools of cell lines from midbrain tissues of 8 weekhuman fetuses were examined, approximately 0.1% of total cells wereconsistently TH-positive dopaminergic neurons, which is also theproportion found in the unmodified stem cell cultures. Clonal analysisrevealed that the TH expression was clonally restricted. That is, amajority of the clones did not contain neurons expressing TH. Of thosethat did, the proportion was variable from one clone to the next.Several pools of cell lines from cortical tissues of 17-20 week oldhuman fetuses were also examined. Interestingly, all of the corticallines gave rise to significantly increased TH-positive neurons comparedto the unmodified stem cells. The proportion of TH-positive neurons was2-4% of the total cells. Subsequently clonal analysis revealed a similarpattern in the distribution of the TH-positive neurons. The majority hadnone, while those that generate TH were present in variable proportions.This pattern had also been observed with clones of unmodified stem cellsfrom several different regions and with several different antigenicmarkers.

Cell lines from spinal cord of 6-10 week old human fetuses were alsoestablished. The pattern of neuronal differentiation was the same asfrom other regions, although their stem cell morphology and growthcharacteristics were distinct.

Thus, the genetic modification of neural stem cells with c-myc does notalter their intrinsic differentiation capacities. In all of the celllines through extensive continues culture periods, no evidence of tumorformation or other abnormal transformation was noted. Upon karyotypeanalysis of one pool of human cortical cell lines at passage 14, anormal diploid chromosome pattern with no aberrant rearrangement wasobserved. Thus, regulation of mitotic capacity by c-myc, which is acellular gene normally present in every eukaryotic cell and a well-knownkey regulator of cell cycle machinery, is not oncogenic and providessignificant advantages over other methods using viral oncogenes such asv-myc or SV40 large T antigen.

-   6. Other Cell Types

The genetic modification with c-myc can be made at any time during theculture period. Since the expression of c-myc itself is not mitogenic,i.e., non-transforming, a culture condition which promotes theproliferation of a particular neural precursor population is aprerequisite. Purified growth factors such as aFGF (acidic fibroblastgrowth factor), bFGF, EFG and TGFα can proliferate a variety ofdifferent neural cell types. Although most of the descriptions abovewere on multipotent CNS stem cells as one predominant population,several different cell types were observed during clonal analysis.

One significant population was a population of bipotential progenitorclones which, upon differentiation, gave rise to neurons and astrocyteswith apparent absence of oligodendrocytes. These bipotential progenitorswere quite similar to the multipotential stem cells in their morphologyduring growth. The differentiation pattern was also similar giving riseto about 50% neurons and 50% astrocytes. Thus, key defining differencebetween the two populations is the absence of oligodendrocytes indifferentiated cultures.

A second cell population arising from c-myc modification of primaryneural cultures was a population of unipotential neuronal progenitorclones which consisted only of neurons. These neuronal progenitor cloneswere of smaller clone size and assumed distinct, immature neuronalmorphologies during proliferation and expressed tau proteins and/orbeta-tubulin III Examples are provided in FIG. 5. Two distinct celltypes were observed (FIG. 5A). One type was small-bodied cells with veryshort single process stemming out from the cell body, which grew intight clusters. These cells were immunoreactive with anti-tau antibodybut not with anti-tubulin IIlb antibody while dividing (FIG. 5B). Theother type of cells were cells with distinctively elongated neuriteswithout extensive branching, which grew in a smaller, more scatteredpattern suggesting higher migratory capacity. In contrast to the firsttype, they were also immunoreactive with both anti-tau antibody and withanti-tubulin lllb antibody (FIG. 5C and 5D, respectively). Often times,the second cell types were-found near or intermixed with the first celltype suggesting that they are two stages of a single continuouslineage-committed neuronal progenitors, with tau+/TuJ1+ state being themore mature state.

A third cell population arising from c-myc-modified neural cell culturewas a population of clones consisting of glia only. Most of these cloneswere astrocytic with little or no oligodendrocytes.

Those results indicate that many neural precursor lineages respondsimilarly to the over-expression of c-myc. In addition to primary neuralcultures prepared from nervous system tissues of mammals, recentadvances in embryonic stem cell cultures indicate that various neuralprecursors form in vitro during differentiation of totipotential orpluripotent embryonic stem cells and cell lines maintained in culturefor long term (Renoncourt et al., Mech. Dev. (1998) 78, 185; Svendsen etal., Trends Neurosci. (1999) 22, 357; Brustle et al., Science (1999)285, 754). These cultures can generate nestin-positive neural precursorcells which can then be transferred to serum-free medium andsubsequently expanded with bFGF and/or EGF for short term. Long-term,mass expansion has not been feasible since the initial neural precursorformation is inefficient. However, by utilizing the genetic modificationmethod with the c-myc gene described here, those transient neuralprecursors may be turned into stable cell lines.

Neural precursors including multipotential neural stem cells can beisolated from adult brains and can be cultured in serum-free conditions.However, this process is inefficient, resulting in only a small numberof proliferative cells. However, with the transfer of c-myc gene asdescribed here, one can establish stable cell lines from such smallnumber of cells obtained from neural tissue biopsies.

c-myc is involved in many different cellular processes such as apoptosisin addition to cell cycle regulation. c-myc has been used previously totransform cells of non-neural origins. However, these previous studieswere done with already stable cell lines such as 3T3 fibroblast celllines and to produce neoplastic state of the cell lines, which had beenselected based on spontaneous chromosomal aberrations which conferredmitogen-independence. Other studies tried to use an immortalizationprocess to turn post-mitotic neurons to re-enter the cell cycle.

CNS stem cells are already mitotic and the mitogenic culture conditionsfor a long-term expansion of up to 30 cell doublings has already beenestablished. Thus, our objective has been to increase the expansioncapacity well beyond the 30 cell doublings at least up to the beginningof senescence which is thought to occur between 60 and 80 cell cycles.Sixty cell doublings represent an 1×10¹⁸-fold increase in cell numberwhich is large enough for screening one million chemical libraries, eachconsisting of 500,000 compounds or large enough to provide celltherapies for 50 billion Parkinson's patients. The key concept has beento find a “gentle” modification of the cells so as not to disrupt theirintrinsic neuronal differentiation capacity while providing an enhancedgrowth capacity under the culture conditions established for primary CNSstem cells.

Increasing the concentration of active c-myc protein leads to thegeneration of stable human CNS stem cell lines. This effect occurs notby overtly deregulating mitotic and differentiation parameters of thecell cycle but by providing resistance to autocrine and paracrinefactors that induce restriction of multipotentiality toward a glialprogenitor state. The consequence is not an oncogenic transformation ofthe cells but rather a stabilization of the cell growth. Thus,endogenous signals which trigger the differentiation, such as thosepresent at confluent cell density, are still effective. The celldivision is still dependent upon the supply of proper exogenous mitogenssuch as bFGF and/or EGF and/or serum. Differentiation of the stem celllines to mature functional neurons is as efficient at the end of the60-cell doublings as in the unmodified primary cells. A variety ofneurotransmitter phenotypes and their relative proportions aremaintained throughout the expansion.

The c-myc activity in these examples was controlled by constructing achimeric protein of c-myc fused to a fragment of estrogen receptorprotein (Eiler et. al., Nature (1988) 340, 60). The intended role of theestrogen is to provide a control over the amount of functionally activec-myc induced in the cell. The estrogen receptor portion of the chimericprotein is activated when it binds with a cell-permeable agonist orantagonist such as estradiol or tamoxifen.

Most members of the nuclear receptor superfamily act similarly in thatcell-permeable ligands diffuse through the plasma bilayer and bind totheir receptor which is then transported to the nucleus as a complex andinduces a variety of transcription related events. The ligand bindingdomain of these nuclear receptor proteins and their ligands cansubstitute for the estrogen receptor and β-estradiol in order toregulate functions of the fused c-myc protein moiety. Examples of suchnuclear receptors are glucocorticoid receptor, progesterone receptor,androgen receptors, vitamin D receptor, thyroid hormone receptors,retinoic acid receptors, and ecdysone receptor. Each of these receptorscan be activated intracellularly by adding to the culture medium itsappropriate ligands. Examples of the ligands are steroid hormones suchas glucocorticoid or dexamethasone, thyroid hormones, retinoids such asretinoic acids, vitamin D, and the insect molting hormone, ecdysone, aswell as their synthetic analogs designed to act on the respectivereceptors. All of these compounds are small, hydrophobic molecules whichcan traverse the cell membrane once supplied extracellularly.

Some receptor-ligand systems are better suited than others for thepurpose of regulating the over-expressed c-myc. For instance, for thepurpose of transplanting cmyc-modified cells into tissues as a treatmentof a disease, it would be desirable that the c-myc-receptor chimericprotein should not respond to endogenous physiological ligands. Thec-myc-estrogen receptor described here has the disadvantage thatpotentially high level of estrogen present in female patients may haveunexpected effects on the cells. In another instance, the ligands usedto control the c-myc activity in culture may have their own unrelatedeffects on endogenous receptors. Thus, an ideal receptor-ligand systemis one in which the receptor moiety of the fusion protein does notrecognize the endogenous ligand and in which the ligand is a syntheticcompound which has no adverse effect on the cells. One such potentialsystem is human progesterone receptor and its antagonist ligand,RU38486. It has been established that the ligand binding fragment of thehuman progesterone receptor does not respond to the endogenous ligand,progesterone but is sensitively activated by a synthetic analog ofprogesterone, RU38486, while RU38486 does not activate the endogenousfull-length progesterone receptor (Wang et al., Proc Natl. Acad. Sci.USA (1994) 91, 8180).

Thus, one enhanced c-myc expression system to produce stable cell lineswould be to construct a plasmid in which the human c-myc gene is fusedto the ligand binding domain of the human progesterone receptor with theC-terminal deletion of 12 amino acids, to cut out the fused DNA(c-mycPR), ligate to the retroviral plasmid, pLXSN, at downstream of 5′LTR, and to generate the intact retrovirus expressing the chimericprotein, c-myc-progesterone receptor (MycPR).

The commercial utilities of the mitotically enhanced CNS stem cells are:cell transplantation of the TH-positive dopaminergic neurons to treatParkinson's disease; substrate for screening potential pharmacologicalcompounds; a reproducible source of gene and protein levels of the cellsinfluenced by a specific agent or protocol designed to represent/mimic adisease process; a reproducible source of novel genes and proteins; areproducible source of neurons and glia for engineering of threedimensional tissues and neural prosthesis; a delivery vehicle ofpotentially therapeutic large molecule compounds such as NGF to treatAlzheimer's disease; and the starting population to further derive invitro various committed neuronal progenitor populations such asproliferative TH-expressing neuronal cells.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not limited to thedisclosed embodiments, but on the contrary is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims. Thus, it is to be understood thatvariations in the present invention can be made without departing fromthe novel aspects of this invention as defined in the claims. Allpatents and articles cited herein are hereby incorporated by referencein their entirety and relied upon.

What is claimed is:
 1. A method for producing a stable cell line ofhuman neural precursor cells in vitro, comprising the steps of: a)preparing a culture of neural precursor cells in a serum-free medium; b)culturing the neural precursor cells in the presence of a first mitogen,wherein said first mitogen is selected from the group consisting ofaFGF, bFGF, EGF, TGFα and combinations thereof; c) introducing into theneural precursor cells a recombinant DNA construct comprising a receptorligand-regulated c-myc gene, wherein the c-myc gene is fused with DNAencoding a ligand-binding domain of a nuclear receptor; d) contactingthe neural precursor cells with an agent that binds the ligand-bindingdomain of the nuclear receptor, wherein the agent comprises amyc-activating chemical selected from the group consisting ofβ-estradiol, RU38486, dexamethasone, thyroid hormones, retinoids, andecdysone; e) further culturing the neural precursor cells in a mediumcontaining the first mitogen and a second mitogen to produce a stablecell line of neural precursor cells, wherein said second mitogen isselected from the group consisting of aFGF, bFGF, EGF, TGFα, serum andcombinations thereof, with the proviso that the second mitogen is otherthan the first mitogen, and f) expanding the neural precursor cells ofthe stable cell line beyond thirty cell doublings in the mediumcontaining the first mitogen and the second mitogen, wherein theexpanded neural precursor cells of the stable cell line of neuralprecursor cells are capable of differentiating into neurons, astrocytes,and oligodendrocytes after removal of the first and second mitogen fromthe medium.
 2. The method of claim 1, wherein the c-myc gene is fusedwith a DNA sequence coding for a ligand-binding domain of a nuclearreceptor selected from the group consisting of a ligand binding domainfor an estrogen receptor, an androgen receptor, a progesterone receptor,a glucocorticoid receptor, a thyroid hormone receptor, a retinoidreceptor, and an ecdysone receptor.
 3. The method of claim 1, whereinthe human neural precursor cells are derived from an in vitro culture ofpluripotent embryonic stem cells.
 4. A cell line produced according tothe method of claim
 1. 5. The cell line of claim 4, wherein the neuralprecursor cells of the produced stable cell line maintain amultipotential capacity beyond thirty cell doublings to differentiateinto neurons, astrocytes and oligodendrocytes.
 6. The cell line of claim4, wherein the neural precursor cells of the produced stable cell linemaintain a bipotential capacity beyond thirty cell doublings todifferentiate into neurons and astrocytes.
 7. The cell line of claim 4,wherein the neural precursor cells of the produced stable cell linemaintain a bipotential capacity beyond thirty cell doublings todifferentiate into astrocytes and oligodendrocytes.
 8. The cell line ofclaim 4, wherein the neural precursor cells of the produced stable cellline maintain a capacity beyond thirty cell doublings to differentiateinto neurons.
 9. The cell line of claim 4, wherein the neural precursorcells of the produced stable cell line maintain a capacity beyond thirtycell doublings to differentiate into astrocytes.
 10. A method forproducing a stable human neural precursor cells clone in vitro,comprising the steps of: a) preparing a culture of neural precursorcells in a serum-free medium; b) culturing the neural precursor cells inthe presence of a first mitogen, wherein said first mitogen is selectedfrom the group consisting of aFGF, bFGF, EGF, TGFα and combinationsthereof; c) introducing into the neural precursor cells a recombinantDNA construct comprising a retroviral vector containing theneomycin-resistance gene and a fusion gene of human c-myc cDNA and humanestrogen receptor cDNA; d) further culturing the neural precursor cellsin a medium containing the first mitogen and a second mitogen, whereinsaid second mitogen is selected from the group consisting of aFGF, bFGF,EGF, TGFα, serum and combinations thereof, with the proviso that thesecond mitogen is other than the first mitogen, wherein the mediumincludes G418 to select for the neural precursor cells containing therecombinant DNA construct, wherein the medium includes β-estradiol; e)plating the neural precursor cells obtained from step d) at a celldensity of 0.5×10⁶ to 1.0×10⁶ cells per 100 mm plate by supplementingthe neural precursor cells with unmodified primary cells in thecontinued presence of G418 in a medium containing the first mitogen andthe second mitogen, and f) expanding the neural precursor cells of thestable cell line beyond thirty cell doublings in the continued presenceof G418 in the medium containing the first mitogen and the secondmitogen, and g) isolating a stable neural precursor clone from theplated neural precursor cells.
 11. The method of claim 10, wherein thehuman neural precursor cells are derived from an in vitro culture ofpluripotent embryonic stem cells.
 12. A cell line produced according tothe method of claim
 10. 13. The cell line of claim 12, wherein theneural precursor cells of the produced stable cell line maintain amultipotential capacity beyond thirty cell doublings to differentiateinto neurons, astrocytes and oligodendrocytes.
 14. The cell line ofclaim 12, wherein the neural precursor cells of the produced stable cellline maintain a bipotential capacity beyond thirty cell doublings todifferentiate into neurons and astrocytes.
 15. The cell line of claim12, wherein the neural precursor cells of the produced stable cell linemaintain a bipotential capacity beyond thirty cell doublings todifferentiate into astrocytes and oligodendrocytes.
 16. The cell line ofclaim 12, wherein the neural precursor cells of the produced stable cellline maintain a capacity beyond thirty cell doublings to differentiateinto neurons.
 17. The cell line of claim 12, wherein the neuralprecursor cells of the produced stable cell line maintain a capacitybeyond thirty cell doublings to differentiate into astrocytes.
 18. Thecell line of claim 12, wherein the neural precursor cells of theproduced stable cell line maintain a capacity beyond thirty celldoublings to differentiate into oligodendrocytes.
 19. The cell line ofclaim 4, wherein the neural precursor cells of the produced stable cellline maintain a capacity beyond thirty cell doublings to differentiateinto oligodendrocytes.